Moloney MSV temperature sensitive transformation mutants have been isolated following UV mutagenesis. Cell clones transformed by mutant viruses were able to form colonies in agar suspension at 34 degrees, and exhibited a transformed morphology and elevated hexose uptake at that temperature as well. At 39 degrees the colony forming ability of the cells was reduced over 1000 fold and the cells exhibited a non-transformed morphology and normal levels of hexose uptake. The mutant virus was rescuable and transferable to other cells, producing the same ts phenotype. Ts transformed cells contain a 58,000 dalton egg-related polyprotein. This protein is phosphorylated at 34 degrees but the level of phosphorylation is drastically reduced at 39 degrees. MSV pseudotypes produced by these cells soon after MLV infection contain a heat-labile phosphokinase. Work is in progress to localize the ts lesion on the MSV genome and to characterize the ts phosphorylation reaction and its substrate.